The research program is focused on the detailed mechanisms underlying the initiation of innate immune responses by the receptors and the ensuing signal transduction pathways. The innate immune system is the first line of defense against infection. It also initiates and directs the proper function of the adaptive immune response, the other branch of the immune system. The potency of the innate immune system has been harnessed as vaccination adjuvants against infections, cancer, and autoimmune diseases. Nonetheless, the underlying mechanisms of action only started to be delineated about ten years ago, when a major family of the pattern recognition receptors (PRRs) was identified. It is now appreciated that several families of PRRs are involved in regulating proper immune responses. Recent crystal structures of the TLR2, TLR3 and TLR4 extracellular domains have provided a framework upon which further investigation of the innate immune recognition can be conducted. It is however apparent from these structural studies that the mechanisms of ligand recognition varies significantly among different PRRs. Our program integrates biochemical studies with extensive structural analysis of membrane bound (such as TLR9) and cytoplasmic (such as RIG-I, NLRP1 and AIM2) receptors, either in complex with their ligands or downstream adapters/effector molecules. A critical feature of these innate immune receptors is that they distinguish among various classes of pathogenic molecules while retaining their capacity for responsiveness to a large number of related structures within a given biochemical class. How the binding domains of the innate receptors achieve such broad reactivity at the atomic level is one of the key issues this project addresses. Such information could be used to guide the development of new therapeutics that can either enhance or limit immune activation involving these receptors. Ligand binding by these receptors in turn initiates molecular signaling cascades that ultimately lead to innate cellular responses that help fight infection and guide the adaptive immune responses. The project aims to decipher this signaling network through studying protein-protein interactions, using X-ray crystallography in conjunction with other biophysical and biochemical techniques. The ultimate goal is to not only delineate the mechanisms of innate immune responses at atomic details and contribute to our general understanding of signal transduction, but also to lay a foundation for future clinical exploitation of the innate immune system for human benefit, such as the development of more effective vaccine adjuvants. In the past year, we have achieved progress in the following area: 1). In order to take full advantage of the bacterial expression system, we have engineered and optimized a series of bacterial expression vectors (based on pET-30) using a pair of restriction enzyme sites (Sal I and Not I) to clone target genes. This bacterial expression platform allows us to conveniently shuttle coding sequences among various protein expression and purification tags, such as His8, GB1, NusA, GST, MBP, Thioredoxin, and Carbonic anhydrase, either at the amino- or carboxyl termini of our target proteins. Most of the engineered vectors also contain TEV protease cleavage site between our protein of interest and expression/purification tags. 2). In light of the difficulties we encountered in scaling up protein expression for various innate receptors such as TLR9 and NLRs, we have established a new unicellular eukaryotic expression system, the Leishmania tarentolae from Jena Bioscience. This is an easy to culture eukaryotic organism that is non-pathogenic to mammals, has fully eukaryotic post-translational modification machinery such as glycosylation, and is amenable to transformation using shuttle vectors modified in E. coli. We have established the Leishmania culture in the lab and performed transformation of TLR constructs in inducible expression vectors. 3). Last year, we determined the crystal structure of the MyD88 TIR domain. We have continued our efforts in studying the mechanisms of TIR domain mediated protein-protein interactions. Recently we have crystallized the TIR domains of TIRAP, a host binding partner for MyD88, and the TIR domain of TcpC, a pathogen protein that targets MyD88 signaling as an immune evasion strategy. We have collected a 2.6 angstrom native dataset for the TIRAP crystals, and are performing MAD data collection with selenomethionine labeled TIRAP. We anticipate determining its structure in the near future and perform studies of TIRAP and TcpC binding with MyD88 both in vitro and in silico. 4). We are continuing our studies of TIR-TIR domain interaction using yeast two-hybrid assays and NMR titration experiments in collaboration with Nico Tjandras group at NHLBI. We have mapped out a number of MyD88 TIR self-dimerization interface residues using yeast two-hybrid assay. Our NMR titration experiments have been hampered by the tendency of TIR domains to aggregate and precipitate at high concentrations. Our recent results show that the binding of TcpC TIR domain with MyD88 is amenable to NMR titration experiments in solution. We will further characterize this binding and correlate with our yeast two-hybrid results. 5). For several years, we met considerable challenges in our efforts to express the RIG-I family of receptors and adapter proteins or binding partners. Recently we succeeded in expressing and purifying RIG-I full-length, RIG-I helicase, RIG-I helicase plus RD domain, MDA5 CARD domain, ATG5, ATG7 and ATG10 proteins as soluble monomers. We are conducting crystallization screens as well as chemical modification strategies to enhance crystallization probability. Using the above purified proteins, we have established a binding assay of RIG-I with RNA or poly I:C. We will use this assay to guide our efforts in producing RIG-I:RNA complexes amenable to crystallization. 6). We have been working on expression of a number of the NLR receptor family members. Recently we determined the crystal structure of the Leucine-rich repeats (LRR) from NLRP1, a NLR family member that is known to trigger inflammation in response to muramyl dipeptide (MDP). MDP is a building block of the cell wall peptidoglycan from both gram positive and gram negative bacteria. The crystal structure and solution studies with Peter Schucks group (NIBIB) both demonstrate a tetramer of the NLRP1 LRR. Each tetramer contains two MDP molecules bound between two LRRs. Interaction of LRR with MDP was confirmed by mass spectrometry and NMR titration experiments, although the apparent affinity appears to be low, suggesting that other domains of the full-length NLRP1 may also contribute to its interaction with MDP. MDP is the minimal component of the Freunds adjuvant, a potent adjuvant with high toxicity. Understanding the mechanisms of MDP binding and stimulation of immune receptors could facilitate the development of effective adjuvants with low toxicity. 7). A new family of cytosolic DNA receptors was recently reported that contain a HIN-200 domain that recognizes DNA and a Pyrin domain that recruits adapter protein ASC to activate caspase-1 and pro IL-1. AIM2 (absent in melanoma 2) is the most intensively studied in this new family. In order to understand its mechanism of DNA recognition, we have expressed and purified the HIN-200 domain of AIM2. The purified protein forms large aggregates in complex with fragments of DNAs from bacteria. We are in the process of manipulating our purification protocols to obtain non-aggregated forms for crystallization.